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APTAMER BIOSENSORS AS DNA/RNA-BASED TOOLS FOR PROTEOMICS
S. Tombelli, M. MasciniUniversità degli Studi di Firenze, Italy
Department of Chemistry
Outline
• Introduction on aptamersdefinitionselection procedurestarget moleculescomparison with antibodies
• Aptamer based biosensorsacoustic sensorsmicromechanical sensorsoptical sensorsarrays
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Aptamers are oligonucleotides (DNA or RNA molecules) that can bind with high affinity and specificity to a wide range of target molecules (proteins, peptides, drugs, vitamins and other organic or inorganic compounds).
Aptamers
They were “discovered” in 1990 by the development of an in vitro selection and amplification technique, known as SELEX (Systematic Evolution of Ligands by Exponential enrichment).
Their name is derived from the Latin word “aptus” which means “to fit” and the Greek suffix “-mer”.
T7 Constantregion
Constantregion
Randomsequence
5’ 3’
A library containing a 40-nucleotide random region is represented by 440 (~1024) individual sequences available for partitioning. Normally, the starting round contains no more than 1014-1015 individual sequences.
Cloning and sequencing
The SELEX process
Targetmolecule
Systematic Evolution of Ligands by Exponential enrichment
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Target molecules
PROTEINSSyrian golden hamster prionEscherichia coli SelBL-selectinTyrosine phosphatase
Ff gene 5ThrombinHIV-1 TatHIV-1 Rev
Inosine monophosphate dehydrogenaseVascular endothelial growth factorBasic fibroblast growth factor
Human IgETaq DNA polymeraseIron regulatory proteinHuman oncostatin M
Human neutrophil elastaseHuman CD4
INORGANIC COMPOUNDSMalachite greenMg2+
ORGANIC COMPOUNDSATP
FMNTheophyllineOrganic dyes
Cocaine
VITAMINSCyanonobalaminBiotin
DRUGSNeomycin B
StretpomycinTobramycinTetracyclinKanamycine A
J.C. Cox, A.D. Ellington, Bioorg. Med. Chem. 9, 2525-2531, (2001)
• PhotoSELEX:modified ssDNA aptamers capable of photocross-linking the target molecule.The method is based on the incorporation of a modified nucleotide, 5-bromo-2’deoxyuridine (BrdU), activated by absorption of light, in place of a native base in the randomised oligonucleotide library. The aptamers selected with this method have the ability to form a photo-induced covalent bond with the target
Automation and modification of the SELEX process
M.C., Golden, B.D. Collins, M.C. Willis, T.H. Koch, J. Biotechnol. 81, 167-178, (2000)C. Bock et al., Proteomics, 4, 609-618, 2004
• Automated selection of aptamers
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Applications based on molecular recognition:
Therapeutics: aptamers have been selected to disrupt the function of their targets and
to inhibit or modify the metabolism associated with that target
Diagnostics: the impressive discrimination between two molecules of very similar structure has suggested that aptamers can be potential diagnostic reagents
Analytical tools: flow cytometrycapillary electrophoresis and electrochromatographyaffinity chromatography
biocomponents in biosensors
Applications
Why aptamer-based biosensors for proteomics?
One major postgenomic goal is the analysis of a chosen cellular proteome
gene expression profiles based on nucleic acid detection
increasing need for rapid and sensitive techniques for direct analysis of proteins
Conventional ligands for non-nucleic acid targets are antibodies
Alternative: aptamersSuccess in the automation of aptamer selection suggests that selection of aptamers on a proteome scale will soon be possible
Profiling arrays based on aptamer biosensors specific for cancer-related molecules will help to profile, at the protein level, the differences between cancer and normal cell types
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Why aptamers can rival antibodies?
• Overcoming of the use of animals for their productionThe immune response can fail when the target molecule, i.e. protein, has a structure similar to endogenous proteins and when the antigen consists of toxic or non-immunogeniccompounds
• After selection, aptamers are produced by chemical synthesis and purified to a very high degree by eliminating the batch-to-batch variation found when using antibodies
• By chemical synthesis, modifications in the aptamer can be introduced enhancing the stability, affinity and specificity of the molecules
• Higher temperature stability
• Because of their small size, denser receptor layers can begenerated
• Amplification by PCR
Transducers
• Acoustic sensors
• Cantilever-based sensors
• Optical sensors
• An almost unexplored area of aptamers is in sensors based on electrochemical detection.Aptamers, being polyanionic, may be attractive for sensing the changes in conductance in the presence, or absence of target binding*.
*K.M. You, S.H. Lee, A. Im, S.B. Lee, Biotechnol. Bioproc. Eng. 8, 64-75, (2003)
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Love-wave biosensor
• Target molecule: Thrombin and Rev peptide• DNA aptamer• Transducer: SAW Love-wave sensor • Immobilisation of the aptamer on the sensor surface:
M.D. Schlensog, T.M.A Gronewold, M. Tewes, M. Famulok, E. Quandt, Sensors Act. B, 101, 308-315, (2004)
s s s s s s s s
Love-wave sensors: highly sensitive analyte detection can be achieved in parallel fashion opening up the possibility of using the sensor-principle in an arrayformat
Love-wave biosensor
• Detection limit72±11 pg/cm2 (thrombin)77±36 pg/cm2 (Rev peptide)
• Dynamic range low nM- low µM
• Affinity-like constant K=500 nM
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Quartz crystal biosensor 1
M. Liss, B. Petersen, H. Wolf, E. Prohaska, Anal. Chem., 74, 4488-4495, (2002)
• Target molecule: human IgE• DNA aptamer compared with anti-IgE antibody• Transducer: quartz crystal microbalance • Immobilisation of the aptamer on the sensor surface: 5’ biotinylated aptamer immobilised on streptavidin fixed on the gold surface with DSP.
Biosensor to detect concentrations and ligand affinity parameters of free unlabeled proteins in real time
• Detection limit 100 µg/L (Ab and aptamer)
• Linear range 0.1-1 mg/L (Ab)0.1-10 mg/L (aptamer)
• Affinity Kd= 1.9 nM (Ab)Kd= 3.6 nM (aptamer)
• Stability crystals modified with aptamers could be stored for several weeks
Quartz crystal biosensor 1
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• Immobilisation of the aptamer on the transducer surface (gold)
Biotinylatedaptamer
(B)
Freeaptamer
Regeneration
(A)
RU
TimeInjection of protein
Washing
Free aptamerTime (sec)
0 500 1000 1500 2000 2500
Freq
uenc
y sh
ift (H
z)
-300
-200
-100
0
100
200
300
400
500
Buffer
Sample
Regeneration
Washing BufferA
B
Quartz crystal biosensor 2Surface Plasmon Resonance biosensor
M. Minunni, S. Tombelli, A. Gullotto, E. Luzi, M. Mascini, Biosens. Bioelectron, in press
HIV-1 Tat protein
HIV-1 replication cycle is controlled by the viral trans-activator of transcription protein Tat (Transcription Trans-Activator).The basic role of Tat is to promote effective elongation of viral mRNA during transcription.
Tat is a small polypeptide of 86-102 amino acids comprising a few functional regions
Acid region Cysteine-richregion
Arginine-richregion
Core region
Glutamine-richregion
1 22 37 48 57 77 101
The arginine-rich region (49-57) of Tat is involved in binding the RNA trans-activation response element (TAR).
TAR is a 59-base hairpin-bulge structure located at the 5’ end of all viral mRNAs.
TAR
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Aptamer for Tat protein
SELEX has been used to isolate an RNA aptamer with high affinity for Tat protein.
The selected aptamer had two repeats of the TAR core binding elements and bound Tat peptide 133 times more efficiently than TAR.*
* R. Yamamoto et al., Genes to Cells (2000) 5, 371.
The selected aptamer RNAtat can be useful for inhibiting the Tat function (as a “decoy”) in vivo and as a diagnostic reagent for the detection of Tat.**
** R. Yamamoto et al., Genes to Cells (2000) 5, 389.
Piezoelectric biosensor results
0
20
40
60
80
100
120
0 0.5 1 1.5 2 2.5 3
conc TAT (ppm)
Shif
t (H
z)
Thermally treated aptamer
Aptamer
Improvements in reproducibility
Non-treated aptamer: CV%=16% (n=3 for each concentration) (1 crystal);CV%=21% (8 crystals)
Thermally treated aptamer: CV%=6% (n=3 for each concentration) (1 crystal); CV%=8% (8 crystals)
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Specificity
-5
0
5
10
15
20
25
freq
uenc
y sh
ift (
Hz)
TAT 0.65 ppmBCL-2 0.65 ppmhIgG 0.65 ppmREV 0.65 ppm
“Blanks”: BSA 0.1% in binding buffer0.2M KCl + 5 mM glutathione (at different dilutions)
0
R2 = 0,997
R2 = 0,975
0
10
20
30
40
50
60
70
80
0 0.2 0.4 0.6 0.8 1 1.2 1.4
conc. TAT (ppm)
Shift
(H
z)
AntibodyAptamer
Comparison with the immunosensor
Anti-Tat antibody(monoclonal)
Y EDAC/NHS
Y
Y
YY
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0
200
400
600
800
1000
1200
1400
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75
Tat conc. (ppm)
Shi
ft (
RU
)
SPR biosensor results (normal aptamer)
Reproducibility
CV%=7% (n=3 for each concentration) (1 crystal);
CV%=6% (8 crystals)
Immobilised AptamerImmobilised Aptamer: ACGAAGCUUGAUCCCGUUUGCCGGUCGAUCGCUUCGA
Selectivity
Response of Rev protein (1.25 ppm): 25% respect to Tat at the same conc.
BSA( 0.1, 0.5 ppm): 0 RU
0
500
1000
1500
2000
2500
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4Conc. Tat (ppm)
Shift
(RU
)
CV% = 3%
StreptavidinBiotin
PolyA(20) tail
aptamer
SPR biosensor results (aptamer with polyA tail)
D.I. Van Ryk, S. Venkatesan, J. of Biol. Chem, 274, 17452-17463
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0
200
400
600
800
1000
1200
1400
1600
1800
Sh
ift (R
U)
Specificity
Tat1.25 ppm 0.125 ppm
Rev1.25 ppm 0.125 ppm Bcl-2
1.25 ppm
hIgG1.25 ppm BSA
1.25 ppm
Cantilever-based biosensor
• Target molecule: Taq DNA polymerase• DNA aptamer• Transducer: cantilever• Immobilisation of the aptamer on the sensor:5’ thiolated aptamer immobilised on gold
Cantilever-based biosensing:
Label-free detectionBatch-fabricatedSmall scale
Arrays can be used in parallel to detect various proteins simultaneously
• Affinity: Kd=15 pM
C.A. Savran, S.M. Knudsen, A.D. Ellington, S.R. Manalis, Anal. Chem. 76, 3194-3198, (2004)
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Fiber-Optic Microarray
• Target molecule: Thrombin
• DNA aptamer• Transducer: optical imaging fibers (fluorescence measurements)
• Immobilisation of the aptamer on the sensor surface: aptamer modified at the 5’ end with an amino group with a spacer arm (C6). The aptamer was fixed onto silica microspheres ((3-aminopropyl)triethoxysilane, glutaraldehyde, polyethylenemine, SBB activated oligonucleotides)
M. Lee, D.R. Walt, Anal. Biochem. 282, 142-146, (2000)
Pivotal role in the blood coagulation and anticoagulation cascades:relevant target protein for drug discovery
• Competitive assay: detection limit of 1 nM
volume of sample 10 µl
dynamic range low nM- low µM
• Stability: the beads modified with aptamer were stable for over 3 months
no degradation in activity during 8 h experiments
Fiber-Optic Microarray
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• Target molecules: thrombininosine monophosphate dehydrogenasevascular endothelial growth factorbasic fibroblast growth factor
Aptamer chip-based biosensor
Cancer-associatedproteins
• DNA and RNA aptamers• Transducer: fluorescence polarisation (anisotropy)• Immobilisation of the aptamer on the sensor surface: 5’ biotinylated aptamers immobilised on streptavidin-derivatised glass substrate.• Aptamers labelled with a single 3’
fluorescein group
T.G. McCauley, N. Hamaguchi, M. Stanton, Anal. Biochem. 319, 244-250, (2003)
• Affinity studies:
solution-phase experiments Kdthrombin= 26 nM
chip-based sensor Kdthrombin= 15 nM
Aptamer chip-based biosensor
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Photoaptamers
C. Bock et al., Proteomics, 4, 609-618, 2004
• Target molecule: 17 target proteins• DNA photoaptamers• Immobilisation of the aptamer on the sensor surface: 5’-amine-photoaptamers spotted onto activated slides
PHOTO-aptamers on the chip
Injection of sample
Gentle washing
Aggressive washing
Conjugation with dye and imaging
Irradiation with UV light(308 nm laser)
Photoaptamers
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• Automation of SELEX will provide the tools for rapid and cheap isolation of new aptamers
• Many details of the highly elaborate genome-based DNA microarray technology are transferred to aptamers chips
• Aptamers could become the perfect complement for proteome-based analyses
Conclusions